Preparation method and application of photoelectrochemical aptamer sensor based on porphyrin COFs

Abstract

The invention relates to the technical field of photoelectrochemical aptamer sensors, and particularly discloses a preparation method of a photoelectrochemical aptamer sensor for rapidly and sensitively detecting beta-amyloid (A beta) protein. A porphyrin-based covalent organic framework (COFs) photoelectrochemical conversion layer is grown in situ on the surface of the electrode through a Schiff base reaction, and after the reaction is finished, the electrode is placed in an activated A beta 42 aptamer solution to be covalently bound through amide; dropwise adding a mercaptohexanol solution on the surface to seal non-specific recognition sites; and finally incubating Abeta42 with different concentrations on the surface of the electrode to obtain the Abeta42 photoelectrochemical aptamer sensor. The Abeta42 photoelectrochemical aptamer sensor can realize rapid and sensitive detection of the content of Abeta42 in a range of 1.0 pmol / L-1. 0mol / L, has the advantages of low background, high selectivity, low cost, simple instrument operation, easy carrying and the like, is expected to play an important role in the field of biomedical analysis and detection, and provides important theoretical and experimental support for diagnosis of Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the technical field of photoelectrochemical aptamer sensors, and in particular to a preparation method of a photoelectrochemical aptamer sensor based on Schiff base reaction and its application in subtype β-amyloid protein Aβ 42 Application in detection. Background Art

[0002] With the continuous growth of my country's economy and significant changes in the social demographic structure, the problem of geriatric diseases brought about by an aging society has received more and more attention, especially neurodegenerative diseases represented by Alzheimer's disease (AD), which has become a major challenge that seriously affects the health and quality of life of the elderly. More and more evidence shows that neurotoxicity induced by Aβ protein aggregation may be the main cause of AD. The aggregation of Aβ protein is a very complex reversible process. Due to the incorrect hydrolysis of amyloid precursor protein (APP) by β-secretase, Aβ monomers rapidly aggregate to form oligomers during the nucleation stage, and metastable oligomers further aggregate into fibrils. The fibrils aggregate into fibers and plaques through secondary nucleation, and accumulate in the interstitium of brain parenchymal cells, leading to a series of cranial nerve dysfunctions. The most common Aβ subtype in the human body is Aβ 40 and Aβ 42 , due to Aβ 42 With two more amino acids and a more compact secondary conformation, Aβ 42 It is more likely to misfold and aggregate, and is more likely to form plaque-like deposits, which are usually more neurotoxic. Studies have shown that Aβ in the plasma of patients with mild AD 42 As the disease progresses, its concentration decreases. 42 The detection not only helps in early diagnosis, but also reflects the progression of the disease.

[0003] Covalent organic frameworks are a type of crystalline organic porous polymers with permanent porosity that are formed by covalently connecting small molecule monomers. They have the characteristics of being pre-designable, uniform and adjustable pore size, and easy functionalization. Due to the diversity of building monomers, topological structures, and connecting bonds, COFs provide a new platform for the structural design and functional development of organic polymers. In the past two decades of development, COFs materials have shifted from structural design to functional design. In recent years, their optical and electrical properties have also been gradually developed. COFs' adjustable pore size and easy connection to functional groups targeting Aβ proteins have shown great advantages in the specific recognition of Aβ proteins.

[0004] Photoelectrochemical (PEC) sensors are an analytical method that combines electrochemistry with photochemistry. They have the advantages of both and can detect substances with or without electrochemical activity. They have attracted much attention due to their low background, high sensitivity, fast and stable response, and no need for sample pretreatment. Due to the different energy forms of the excitation source (light) and the detection signal (electricity), the PEC sensing platform has higher sensitivity and signal-to-noise ratio than traditional electrochemical sensing methods. In addition, the electronic readout system of the PEC sensing platform makes it simple and easy to miniaturize, while optical bioanalysis requires complex and expensive equipment. Highly sensitive PEC biosensors can achieve low-level detection of Aβ protein.

[0005] The present invention was funded by the Hubei Natural Science Foundation (2024AFB496), the Hubei Three Gorges Laboratory Open Fund (SK240006), the Enshi Prefecture "Qihang Special" Science and Technology Plan Project (Research on the Performance of Photoelectric Responsive Covalent Organic Framework Materials), and the Hubei University for Nationalities In-school Research Project (BS24057, XN24034). A real-time monitoring method for Aβ 42 The photoelectrochemical aptamer sensor has a simple preparation method and excellent performance, and is expected to play an important role in the field of biomedical analysis and detection. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a photoelectrochemical aptamer sensor based on porphyrin COFs and its application in Aβ 42 Applications in protein monomer identification and detection.

[0007] In the first aspect, the present invention provides a method for real-time monitoring of Aβ based on porphyrin COFs. 42 The preparation method of the photoelectrochemical aptamer sensor of the protein monomer comprises the following steps:

[0008] S1. Using two symmetrical organic small molecules containing two aldehyde groups and four amino groups as building monomers, COFs thin films are in situ grown on the surface of indium tin oxide (ITO) or fluorine-doped tin oxide (FTO) electrode by Schiff base condensation reaction under the catalysis of Lewis acids, such as acetic acid, aspartic acid, hydroxyproline, proline, etc., to form a photoelectric conversion layer to obtain a modified electrode COFs / M, where M is ITO or FTO;

[0009] S2. Activation of carboxyl-modified Aβ by coupling agent EDC / NHS mixed solution 42 Aptamer Apt solution, placing the COFs / M modified electrode obtained in S1 in the activated Apt solution, the amino group at the end of COFs and the carboxyl group modified by Apt are covalently bonded through an amide bond to obtain an Apt / COFs composite film modified electrode Apt / COFs / M;

[0010] S3. Add a blocking agent to the surface of the modified electrode Apt / COFs / M to block the nonspecific recognition sites. After the blocking is completed, the composite electrode MCH / Apt / COFs / M is obtained, which can detect Aβ rapidly and sensitively. 42 Photoelectrochemical aptasensors.

[0011] Furthermore, before the COFs film is synthesized in situ on the electrode in step S1, the electrode is pretreated, specifically, the ultrasonic electrode is washed alternately with anhydrous ethanol and ultrapure water.

[0012] Furthermore, the organic small molecules in step S1 are 2,5-dihydroxyterephthalaldehyde DHTP and 5,10,15,20-tetrakis(4-aminophenyl)porphyrin TAPP, and further, the molar ratio of DHTP to TAPP is 2:1. Preferably, the amino group of TAPP reacts with the aldehyde group of DHTP to form a Schiff base reaction, and the reaction time is 3 hours.

[0013] Furthermore, in the step S1, a solvent is added during the Schiff base condensation reaction, and the solvent is a mixed solvent of chloroform and dichloromethane (preferably chloroform and dichloromethane in a volume ratio of 3:1).

[0014] Furthermore, in the EDC / NHS mixed solution of coupling agent for activating Apt in step S2, the molar concentration ratio of EDC and NHS is 1:1, and the preferred concentrations are both 100 mmol / L.

[0015] Furthermore, in step S2, the concentration range of Apt is 0.5-3.0 μmol / L (optimal concentration is 2 μmol / L), and the culture time range is 0.5-4 h (optimal time is 3 h).

[0016] Furthermore, the blocking agent for nonspecific adsorption in step S3 is 6-mercaptohexanol MCH, the concentration of which is 2 mmol / L, and the blocking time is 30 min.

[0017] In a second aspect, the present invention also provides a method for rapid and sensitive detection of Aβ 42 The photoelectrochemical aptamer sensor of protein monomer is prepared by adopting the preparation method.

[0018] In a third aspect, the present invention also provides the above-mentioned photoelectrochemical aptamer sensor for detecting Aβ 42 Application of protein monomer concentration, through Aβ 42 Quantitative analysis of Aβ by the difference between the photocurrent and dark current of protein-modified sensors 42 Protein monomer concentration, if the sample contains Aβ 42 protein monomers, the photocurrent response will decrease, thus 42Qualitative analysis of protein monomers; the magnitude of the photocurrent response decrease is related to the Aβ content in the sample 42 The concentration of protein monomers is proportional to the Aβ 42 Protein monomers were quantitatively analyzed.

[0019] Furthermore, the photoelectrochemical aptamer sensor detects Aβ 42 The specific steps for protein monomer concentration are:

[0020] (1) Different concentrations of Aβ were drop-coated on the photoelectrochemical aptamer sensor. 42 Protein monomer solution;

[0021] (2) Using a saturated silver chloride electrode as the reference electrode and a platinum wire electrode as the counter electrode, Aβ 42 Modify the MCH / Apt / COFs / ITO electrode as the working electrode to construct a three-electrode system;

[0022] (3) placing the three-electrode system in a phosphate buffer solution;

[0023] (4) With 0 V as the bias potential, without additional adjustment of electron donor / acceptor, the current-time curve was measured under alternating light / closed conditions to calculate the Aβ concentration at different concentrations 42 The photocurrent response value of protein monomer is lower than that without Aβ 42 The difference when the protein is a monomer was used to fit a linear curve;

[0024] (5) The different concentrations of Aβ in (1) were added 42 The protein monomer is replaced with the Aβ-containing 42 The photocurrent response value calculated by (1)-(4) above is less than that without Aβ 42 After the difference of protein monomer, the Aβ-containing 42 Aβ in solution of protein monomers 42 Protein monomer concentration.

[0025] The prepared photoelectrochemical aptasensor was applied to Aβ 42 The linear range of protein monomer detection is wide, from 1.0 pmol to 1.0 μmol / L. Within this concentration range, the sensitivity of the sensor is 0.6470 μA / (pmol / L) and the detection limit is 0.8131 pmol / L.

[0026] Compared with the prior art, the advantages and beneficial effects of the method of the present invention are as follows:

[0027] (1) Rapid and sensitive detection of Aβ by the present invention 42The photoelectrochemical aptamer sensor of protein monomers is an in-situ synthesis of MCH / Apt / COFs / ITO composite film on the surface of ITO electrode. This composite film is a new type of sensing film, which is prepared for the first time and used for Aβ 42 Protein monomer photoelectrochemical aptamer sensor;

[0028] (2) Rapid and sensitive detection of Aβ by the present invention 42 The preparation method of the photoelectrochemical aptamer sensor of protein monomers was used to prepare the MCH / Apt / COFs / ITO photoelectrochemical aptamer sensor by using a simple Schiff base condensation method and an amide bond covalent bonding method. Scanning electron microscopy characterization showed that the surface of MCH / Apt / COFs / ITO was uneven, the specific surface area of ​​the electrode was large, and the photoelectric conversion efficiency was high, which was beneficial to improve the sensitivity of the sensor. The experimental results showed that the prepared photoelectrochemical aptamer sensor had the advantages of wide linear range, high sensitivity, low detection limit, short response time, high selectivity, good stability and reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The invention provides a rapid and sensitive method for detecting Aβ 42 Roadmap for the preparation of photoelectrochemical aptamer sensors based on protein monomers;

[0030] Figure 2 A roadmap for the synthesis of TAPP / DHTP-COFs;

[0031] Figure 3 Scanning electron microscope (SEM) characterization image of the surface of COFs / ITO modified electrode;

[0032] Figure 4 is the Fourier transform infrared (FT-IR) spectrum of COFs;

[0033] Figure 5 The photocurrent response curves of different modified electrodes;

[0034] Figure 6 It is the photoelectric response current curve of different concentrations of Apt;

[0035] Figure 7 The photoelectric response current curve of Apt at different incubation times;

[0036] Figure 8 The prepared MCH / Apt / COFs / ITO modified electrode reacts with different concentrations of Aβ 42 Time-current curve diagram (linear diagram);

[0037] Fig. 9 Aβ 42Stability curve of protein monomer photoelectrochemical aptamer sensor;

[0038] Fig.10 Aβ 42 Protein monomer photoelectrochemical aptamer sensor selectivity bar graph;

[0039] Fig.11 Aβ 42 Reproducibility bar graph of protein monomer photoelectrochemical aptamer sensor. DETAILED DESCRIPTION

[0040] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solution of the present invention. Obviously, the described embodiments are only part of the implementation of the present invention, not all of the implementations. Based on the implementation of the present invention, all other implementations obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] In the following examples, the indium tin oxide conductive glass ITO electrode used was purchased from South China Xiangcheng Technology Co., Ltd. (size: 40 mm×10 mm×1 mm). Apt was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.

[0042] Example 1

[0043] A rapid and sensitive method for detecting Aβ 42 Preparation method of photoelectrochemical aptamer sensor of protein monomer, such as Figure 1 As shown, the following steps are included:

[0044] S1. At room temperature (25°C), COFs modified ITO electrode (COFs / ITO) was synthesized in situ on ITO electrode by Schiff base condensation reaction with 2,5-dihydroxyterephthalaldehyde (DHTP) and photosensitizer (5,10,15,20-tetrakis(4-aminophenyl)porphyrin (TAPP)) as building monomers under the catalysis of acetic acid (HAc). The reaction route is as follows: Figure 2 As shown;

[0045] S2, the COFs / ITO modified electrode surface obtained in S1 was drop-coated with 2.0 μmol / L activated Aβ 42 The aptamer Apt solution, the amino group at the end of COFs and the carboxyl group modified by Apt are covalently bonded through an amide bond to prepare an Apt / COFs composite film modified ITO electrode (Apt / COFs / ITO);

[0046] S3. MCH is added to the surface of the modified electrode Apt / COFs / ITO obtained in S2 to block the nonspecific recognition sites. After the blocking is completed, the composite electrode MCH / Apt / COFs / ITO is obtained to detect different concentrations of Aβ42 Protein monomer, i.e. rapid and sensitive detection of Aβ 42 Photoelectrochemical aptamer sensor of protein monomer; wherein the MCH concentration is 2mmol / L, the addition amount is 20μL / ITO, and the blocking time after the addition is 30min.

[0047] S4. Coat 20 μL of Aβ on the surface of the prepared modified electrode MCH / Apt / COFs / ITO. 42 Protein, kept wet to incubate with different concentrations of Aβ 42 Protein monomers to obtain different concentrations of Aβ 42 / MCH / Apt / COFs / ITO was placed in PBS solution and the current-time curve was tested under LED white light (40 mW) to obtain the photoelectric response curve.

[0048] It should be noted that S1 in Example 1 specifically includes: placing ITO in a centrifuge tube with a volume of 7 mL, adding 2,5-dihydroxyterephthalaldehyde DHTP and photosensitizer 5,10,15,20-tetrakis (4-aminophenyl) porphyrin TAPP, solvent, and acetic acid thereto, the mass of TAPP and DHTP used for in-situ growth on the surface of the ITO electrode is 0.84 mg and 0.40 mg respectively, the solvent is a mixed solution of chloroform and dichloromethane (volume ratio is 3:1), the volume of the mixed solution is 1400 μL, and the amino group of TAPP reacts with the aldehyde group of DHTP to undergo Schiff base reaction, and the reaction time is 3 hours. The TAPP and DHTP used were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and chloroform and dichloromethane were from China Pharmaceutical Group Co., Ltd.; the ITO electrode of the in-situ grown COFs film is one side with an indium tin oxide conductive film, the range is 10 mm × 10 mm, and the area is 1 cm 2 The concentration of catalyst acetic acid is 10 mol / L, and the volume added to each centrifuge tube is 10 μL. The in-situ grown DHTP / TAPP-COFs film is more uniform, thus having better photoelectric response performance and improving the detection sensitivity of the sensor.

[0049] In Example 1, S2 specifically includes: In Example S1, the coupling agent for activating Apt is an EDC / NHS mixed solution, wherein the concentration of EDC is 100mmol / L, the concentration of NHS is 100mmol / L, 20μL of the coupling agent EDC / NHS mixed solution activates 20μL of 2μmol / L Apt, and the activation time is 30min. The concentration of Apt is 2μmol / L, and the incubation time is 3h, thus obtaining an Apt / COFs composite film modified ITO electrode (Apt / COFs / ITO).

[0050] In Example 1, S3 specifically includes: applying 20 μL of 2 mmol / L mercaptohexanol MCH dropwise to the surface of Apt / COFs / ITO obtained in S2 to block nonspecific recognition sites, thereby preparing a composite electrode MCH / Apt / COFs / ITO, i.e., a photoelectrochemical aptamer sensor. The π-conjugated structure and excellent stability of COFs make the COFs / ITO electrode have good photoelectric conversion efficiency, thereby shortening the Aβ 42 The response time of the protein monomer photoelectrochemical aptamer sensor. In the construction of the photoelectrochemical aptamer sensor, based on Aβ 42 Apt with customized amino acid sequence was used as electrode modifier to improve the selectivity of the sensor.

[0051] In Example 1, S4 specifically includes: PBS solution concentration of 0.1 μmol / L, pH of 7.40, and volume of 20 μL. The electrolytic cell used in the test has a volume of 5 mL and a light window of 1 cm. 3 The light used in the test is LED white light, with a wavelength range of 400-700nm, and the light on / off time is 30s / 20s alternately.

[0052] It should be noted that, before the in-situ synthesis of the DHTP / TAPP-COFs film on the surface of the ITO electrode in S1 in Example 1, the process also includes: pre-treating the ITO electrode, specifically, cleaning the ITO electrode in an ultrasonic cleaner using anhydrous ethanol and ultrapure water alternately, and drying the electrode after ultrasonic cleaning at room temperature for later use.

[0053] Example 1: A DHTP / TAPP-COFs film was generated on an ITO electrode by an in situ synthesis method. The amino group at the end of COFs was covalently bonded to the carboxyl group modified with Apt by an amide method. Finally, MCH was added dropwise to block the nonspecific recognition sites of the modified electrode Apt / COFs / ITO to prepare a new type of Aβ 42 Protein monomer photoelectrochemical aptamer sensor, the Aβ 42 Protein monomer photoelectrochemical aptamer sensors have excellent properties such as wide linear range, high sensitivity, low detection limit, short response time, high selectivity, good stability and reproducibility.

[0054] Performance Testing

[0055] 1. SEM characterization of COFs modified electrode surface

[0056] The surface morphology of the COFs-modified ITO electrode in Example 1 was characterized by SEM technology. Figure 3 shown.

[0057] from Figure 3It can be seen that a very uniform autumn leaf-shaped film structure is clearly visible on the surface of the COFs-modified ITO electrode. The entire surface is uneven and has a large specific surface area. Therefore, the uneven surface structure of the COFs-modified ITO electrode gives it a higher photoelectric conversion efficiency and better enrichment ability, thereby increasing the number of exposed amino groups on the surface, which can connect with more aptamers and further increase its affinity for Aβ 42 Sensing sensitivity of protein monomers.

[0058] 2. FT-IR characterization of COFs materials

[0059] The chemical structure of the COFs grown in situ in a centrifuge tube according to the synthesis method S1 in Example 1 was characterized by Fourier transform infrared spectroscopy to evaluate whether imine condensation between the amino group and the aldehyde group occurred. Figure 4 shown.

[0060] from Figure 4 It can be seen that the COFs material grown in situ in Example 1 has a -1 There is an obvious characteristic peak of -C=N- bond, indicating that Schiff base condensation reaction successfully occurred between the amino group of TAPP and the aldehyde group of DHTP.

[0061] 3. Photoelectrochemical responses on different modified electrodes

[0062] The COFs / ITO modified electrode prepared in Example 1S1, the Apt / COFs / ITO modified electrode prepared in Example 1S2, and the Aβ prepared in Example 1S4 were tested respectively. 42 Aβ at a concentration of 1 nmol / L 42 The photoelectrochemical response of the / MCH / Apt / COFs / ITO modified electrode is shown in the following figure. Figure 5 shown.

[0063] The specific test method is: using saturated silver chloride electrode as reference electrode, platinum wire electrode as counter electrode, Aβ 42 / Apt / COFs / ITO or Apt / COFs / ITO modified electrode was used as the working electrode to construct a three-electrode system; 0.1 mol / L phosphate buffer solution with a pH value of 7.40 was used as the base solution, and the current-time curve was tested at a bias voltage of 0 V to evaluate the COFs / ITO modified electrode, Apt / COFs / ITO modified electrode and Aβ 42 Photoelectrochemical response on / MCH / Apt / COFs / ITO modified electrode. Figure 5The results show that the photoelectrochemical response of COFs / ITO modified electrode is very good, and the photocurrent response is about 11.11μA. On the Apt / COFs / ITO modified electrode, due to the non-conductivity of Apt, the electron transport is slowed down, and its photocurrent response is about 7.01μA. 42 After the protein monomers were covalently linked, the protein further slowed down the electron transfer, and the photocurrent response further decreased to about 5.23μA. 42 Protein monomers also have different degrees of delay in electron transfer, so the sensor can realize the Aβ in the sample 42 Detection of protein monomers.

[0064] 4. Effects of Apt concentration and incubation time on photoelectric response

[0065] According to the method in Example 1S2, the concentration of Apt solution was changed to 0.5 μmol / L, 1 μmol / L, 1.5 μmol / L, 2 μmol / L, 2.5 μmol / L and 3 μmol / L, and then the composite electrode Apt / COFs / ITO was prepared according to the same method as Example 1, and the photoelectric response effect of the photoelectrochemical aptamer sensor prepared with different concentrations of Apt was tested. The results are as follows: Figure 6 As shown. Figure 6 It can be seen that when the concentration of Apt is 2 μmol / L, the photocurrent decrease trend of the sensor decreases and gradually reaches stability, so the optimal concentration of Apt is 2 μmol / L.

[0066] According to the method in Example 1S2, the incubation time was changed to 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h and 4h, and then the composite electrode Apt / COFs / ITO was prepared according to the same method as Example 1. The photoelectric response effect of the photoelectrochemical aptamer sensor prepared by Apt at different incubation times was tested. The results are as follows: Figure 7 As shown. Figure 7 It can be seen that when the incubation time of Apt is 3h, the decreasing trend of the sensor photocurrent decreases and gradually reaches stability, so the optimal incubation time of Apt is 3h.

[0067] 5. Aβ 42 Linear range, sensitivity and detection limit of protein monomer photoelectrochemical aptamer sensor

[0068] The current-time curves were tested under alternating light and dark conditions to evaluate the effect of the MCH / Apt / COFs / ITO modified electrode prepared in Example 1S3 on Aβ 42The quantitative analysis capability of protein monomers, i.e., the MCH / Apt / COFs / ITO modified electrode prepared in Example 1 for different concentrations of Aβ 42 The photoelectric response of the protein monomer is reduced. Figure 8 shown.

[0069] The specific experimental method is as follows: according to the description in S4 of Example 1, the prepared MCH / Apt / COFs / ITO modified electrode surface is further incubated with different concentrations of Aβ 42 The protein monomer was placed in 5mL of 0.1mol / L pH=7.40 phosphate buffer solution at a bias voltage of 0V. During the amperometric response, light (LED white light, wavelength range 400-700nm) and dark environment were alternately provided. Specifically, 30s (light) and 20s (darkness) were alternately performed, and the current-time curve test was performed. The difference between the current under light and the current under dark conditions was calculated to evaluate its photoelectric response, and the current of different concentrations of Aβ was calculated. 42 The photocurrent response value of protein monomer is lower than that without Aβ 42 The difference in protein monomers is fitted with a linear curve, such as Figure 8 The data show that with the increase of Aβ 42 As the concentration of protein monomers increased, the difference in reduction gradually increased, from 0.57μA (corresponding to 1.0pmol / L Aβ 42 protein monomer) increased to 4.40μA (corresponding to 1.0μmol / L Aβ 42 protein monomer). Figure 8 It can be seen that in the concentration range of 1.0pmol / L to 1.0μmol / L, the difference in photocurrent response is consistent with Aβ 42 The logarithm of the protein monomer concentration showed a good linear relationship, that is, ΔI (μA) = 0.6470logc (pmol / L) + 0.5311, R 2 =0.9991, the sensitivity of the sensor in this concentration range is 0.6470μA / (pmol / L) (the slope of the linear equation), and the detection limit is 0.8131pmol / L. The detection limit is calculated according to the formula D=3N / S, where D is the detection limit, N is the noise signal (6.98μA), and S is the sensitivity. The results show that the prepared Aβ 42 Protein monomer photoelectrochemical aptamer sensors have the advantages of wide linear range, high sensitivity and low detection limit.

[0070] 6. Aβ 42 Stability, selectivity and reproducibility of protein monomer photoelectrochemical aptamer sensors

[0071] Stability is an important evaluation index of biosensors. The photoelectrochemical aptamer sensor prepared by the present invention can effectively inhibit the occurrence of Aβ in the presence of 10 pmol / L. 42 After incubation in the protein monomer, the test was performed according to the 5 conditions, and the photocurrent response under on / off illumination was recorded 14 times. The results are as follows Fig. 9 As shown, the sensor maintained a stable photocurrent response under multiple cycles (680s) with a relative standard deviation of 7.7%, proving that the sensor has good stability.

[0072] There are many factors that affect the detection of Aβ in the body. 42 Therefore, based on the detection in 5, the photoelectrochemical aptamer sensor in Example 1 was studied for Aβ by adding various interferents. 42 Specifically, the Aβ prepared in Example 1 was studied using the current-time curve. 42 The MCH / Apt / COFs / ITO modified electrode prepared in Example 1 was further incubated with 1.0 nmol / L glucose, dopamine, insulin and Aβ 42 Fibrils replace 100 pmol / L Aβ 42 The protein monomer was placed in 5 mL of 0.1 mol / L pH=7.40 phosphate buffer solution at a bias voltage of 0 V. During the amperometric response, light and dark environments (30 s light and 20 s dark) were provided alternately. The current-time curve test was performed and the difference between the current under light and the current under dark conditions was calculated to evaluate its photoelectric response. Fig.10 As shown in the figure, common interfering factors in biological samples such as dopamine, glucose, and insulin have an impact on Aβ 42 The photocurrent effects of the protein monomers were 5.4%, 1.5% and 11% respectively. The experimental results show that the Aβ prepared in Example 1 42 The protein monomer photoelectrochemical aptamer sensor has good selectivity for Aβ 42 Aβ protein monomers 42 The fiber body also has a certain selectivity (photocurrent influence 54%).

[0073] Reproducibility is also an important reference performance of the sensor. Five MCH / Apt / COFs / ITO modified electrodes prepared in Example 1 were incubated with 1.0 pmol / L Aβ 42 Protein monomers were tested in parallel under the same conditions, and the relative standard deviation of the test results was 9.1% ( Fig.11 ). The results showed that the photoelectrochemical aptamer sensor was effective for Aβ 42The determination of protein monomers has good reproducibility.

[0074] The present invention adopts the method of in situ condensation, covalent linkage and aptamer specific recognition to prepare Aβ 42 / MCH / Apt / COFs / ITO photoelectrochemical aptamer sensor. Scanning electron microscopy characterization shows that the COFs surface is uneven and leaf-like, the electrode has a large specific surface area, and the photoelectric conversion efficiency is high. This application optimizes the detection conditions of the photoelectrochemical aptamer sensor, such as the concentration of Apt and the incubation time. The results show that the Aβ 42 Protein monomer photoelectrochemical aptamer sensors have the properties of wide linear range, high sensitivity, low detection limit, high selectivity, good stability, etc. They have good selectivity for common interfering biological molecules such as glucose, dopamine, insulin, etc. 42 The fibrous body also has a certain degree of recognition ability.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A real-time monitoring of Aβ based on porphyrin COFs 42 The preparation method of the photoelectrochemical aptamer sensor of the protein monomer comprises the following steps: S1. Using two symmetrical organic small molecules containing two aldehyde groups and four amino groups respectively as building monomers, COFs thin films were in situ grown on the surface of indium tin oxide (ITO) or fluorine-doped tin oxide (FTO) electrodes through Schiff base condensation reaction under the catalysis of Lewis acid to form a photoelectric conversion layer to obtain a modified electrode COFs / M, where M is ITO or FTO; S2. Activation of carboxyl-modified Aβ by coupling agent EDC / NHS mixed solution 42 Aptamer Apt solution, placing the COFs / M modified electrode obtained in S1 in the activated Apt solution, the amino group at the end of COFs and the carboxyl group modified by Apt are covalently bonded through an amide bond to prepare an Apt / COFs composite film modified electrode Apt / COFs / M; S3. Add a blocking agent to the surface of the modified electrode Apt / COFs / M to block the nonspecific recognition sites. After the blocking is completed, the composite electrode MCH / Apt / COFs / M is obtained, which can detect Aβ rapidly and sensitively. 42 Photoelectrochemical aptasensors.

2. The preparation method according to claim 1, characterized in that: The Lewis acid is any one of acetic acid, aspartic acid, hydroxyproline and proline.

3. The preparation method according to claim 1, characterized in that: The organic small molecules in step S1 are 2,5-dihydroxyterephthalaldehyde DHTP and 5,10,15,20-tetrakis(4-aminophenyl)porphyrin TAPP.

4. The preparation method according to claim 1, characterized in that: In the step S1, a solvent is added during the Schiff base condensation reaction, and the solvent is a mixed solvent of chloroform and dichloromethane.

5. The preparation method according to claim 1, characterized in that: In the EDC / NHS mixed solution of coupling agent for activating Apt in step S2, the molar concentration ratio of EDC to NHS is 1:

1.

6. The preparation method according to claim 1, characterized in that: The concentration range of Apt in step S2 is 0.5 ~ 3.0 µmol / L, and the incubation time range is 0.5 ~ 4 h.

7. The preparation method according to claim 1, characterized in that: The blocking agent for nonspecific adsorption in step S3 is 6-mercaptohexanol MCH, the concentration of which is 2 mmol / L, and the blocking time is 30 min.

8. The photoelectrochemical aptamer sensor prepared by any one of claims 1 to 7 is used for detecting Aβ 42 Application of protein monomer concentration.

9. The use according to claim 8, characterized in that: The photoelectrochemical aptasensor detects Aβ 42 The specific steps for protein monomer concentration are: (1) Different concentrations of Aβ were drop-coated on the photoelectrochemical aptamer sensor. 42 Protein monomer solution; (2) Using a saturated silver chloride electrode as the reference electrode and a platinum wire electrode as the counter electrode, Aβ 42 Modify the MCH / Apt / COFs / ITO electrode as the working electrode to construct a three-electrode system; (3) placing the three-electrode system in a phosphate buffer solution; (4) With 0 V as the bias potential, without additional adjustment of the electron donor / acceptor, the current-time curve was measured under alternating light-on / off conditions to calculate the Aβ concentration at different concentrations. 42 The photocurrent response value of protein monomer is lower than that without Aβ 42 The difference when the protein is a monomer was used to fit a linear curve; (5) Add different concentrations of Aβ in (1) 42 The protein monomer is replaced with the Aβ-containing 42 The photocurrent response value of the protein monomer solution was calculated by the above (1)-(4) and compared with that without Aβ 42 After the difference of protein monomer, the Aβ-containing 42 Aβ in solution of protein monomers 42 Protein monomer concentration.

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